Brunhilde Knöll

Melatonin suppresses reactive oxygen species induced by UV irradiation in leukocytes

Abstract  An investigation of the antioxidative UV protective effect of melatonin was performed in an in vitro irradiation model with leukocytes. Leukocytes were isolated from EDTA‐treated whole blood and taken up in phosphate‐buffered saline (PBS). Five of 10 aliquots were incubated with 2 mmol/L melatonin and 5 with PBS as a control. The samples were irradiated by UV light (280–360 nm, max: 310 nm) at doses between 75 and 300 mJ/cm2 or left unirradiated. Radical formation was measured using the chemiluminescence technique. Staining with trypan blue was performed to assess cell viability. Melatonin significantly suppressed radical formation in cell solutions irradiated from 75 to 300 mJ/cm2 (P ≤ 0.001). Controls showed an increase of reactive oxygen species (ROS) formation as a sign of oxidative stress when irradiated with increasing UV doses and a maximum ROS formation under 300 mJ/cm2 UV light. The cytotoxicity of UV light was reduced by melatonin up to a UV dose of 1.5 J/cm2. Leukocytes were suitable cells for the evaluation of the efficacy of melatonin as a radical scavenger under UV light. The results confirm that the clinically observed UV protective effects of melatonin may be at least partially based on its radical scavenging properties.

Melatonin reduces UV-induced reactive oxygen species in a dose-dependent manner in IL-3-stimulated leukocytes

Reactive oxygen species (ROS) are presumed to be involved in inflammatory UV reactions of the skin. This in vitro study was performed to investigate the suppressive effect of melatonin in interleukin‐3 (IL‐3) stimulated leukocytes. Neutrophilic granulocytes were isolated from EDTA‐treated whole blood and placed in a phosphate‐buffered saline (PBS) containing IL‐3. Cell suspensions were either treated with PBS (control) or with increasing doses of melatonin (0.1, 0.5, 1, 2, 3, 5, 7.5, 10 mmol). One PBS solution was left unirradiated and the other nine solutions (PBS and melatonin) were irradiated with 750 mJ/cm2 UVB light (280–360 nm, max: 310 nm). Radical formation was measured by the chemiluminescence technique. UV‐irradiated leukocytes showed a 5‐fold higher radical formation than unirradiated leukocytes. Melatonin, in increasing doses in powers of ten, led to a maximum suppression of free radicals at 10 nmol (P=0.01) and 1 mmol melatonin (P=0.001), showing a biphasic, non‐linear, dose–response relationship. Melatonin, given in amounts of 0.1–10 mmol, led to a direct dose‐dependent suppression of ROS. Radical formation was suppressed significantly in a range from 0.5 to 10 mmol (P=0.001). Melatonin is known to function as a radical scavenger and antioxidant; some of these melatonin effects may be receptor independent, while others may be receptor dependent.

Melatonin Suppresses Reactive Oxygen Species in UV-Irradiated Leukocytes More than Vitamin C and Trolox

To prove the relative potency of melatonin as a radical scavenger in UV-irradiated leukocytes, it was compared to other antioxidative substances such as trolox and vitamin C. Human leukocytes were isolated from EDTA whole blood and incubated with melatonin, trolox and vitamin C. The experiments were performed in a wide concentration range from 0.1 nM to 1 mM and in a small concentration range from 0.5 to 2 mM (mel), 5 mM (trolox) and 10 mM (vit. C). Irradiation was performed with UV-light (280-360 nm) at a dose of 750 mJ/cm2. Radical formation was measured by the chemiluminescence technique. The maximum effect of radical suppression was seen at a concentration of 10 nM (p = 0.003) and 1 mM melatonin (p < 0.001) and vitamin C (p = 0.002; p < 0.001), respectively. ROS suppression by trolox was only significant at 1 mM (p < 0.001). In the small concentration range, a linear dose-response relationship was found and melatonin showed the strongest radical suppression (IC50 = 0.21 mM) followed by vitamin C (IC50 = 0.26 mM) and trolox (IC50 = 1.03 mM).

Serum protein S100β in patients with malignant melanoma detected by an immunoluminometric assay

Abstract S100 protein is well established as a diagnostic tool in malignant melanoma immunohistology. In this study we measured S100β in serum with a recently developed luminometric immunoassay with a detection limit of 0.02 μg/l. By measuring S-100β in a group of apparently healthy individuals a mean value of 0.031 ± 0.026 μg/l was found. In the reference group, serum S100β was below 0.12 μg/l in all cases. To assess the sensitivity of the assay we investigated serum S-100β levels in 371 serum samples of 315 patients with histological proven malignant melanoma at different disease stages. Staging was performed according to the German Society of Dermatology classification. Significant differences were observed between the control group and stages IIb (P = 0.01) and IV (P = 0.001). In tumour-bearing patients of stages IIIb and IV, the difference was highly significant (P < 0.0001). S100β > 0.20 μg/l helps to distinguish between tumour-free and tumour-bearing patients with a specificity of 97.0% and a sensitivity of 64.6%. Our results demonstrate the serum S100β is of limited value as a melanoma marker. However, it has clinical significance for identifying tumour-positive patients in advanced malignant melanoma stages III and IV.

Serum matrix metalloproteinase-2 in patients with malignant melanoma.

Abstract. Purpose: Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases that are able to degrade extracellular matrix components. MMPs play a role in tumor invasion and tumor metastasis. MMP-2 (also known as gelatinase A) is expressed in human melanoma cells. Methods: In this study, we measured MMP-2 in 337 serum probes of 166 melanoma patients with a recently developed enzyme immunoassay and compared these data with the tumor stage, presence of metastases, and the levels of S100beta and soluble intracellular adhesion molecule-1 (sICAM-1) in serum. Results: The mean levels were (189.2±50.8) ng/ml for MMP-2, (263.2±74.1) ng/ml for sICAM-1, and (0.424±1.568) U/ml for S100beta. There was a statistical significant correlation of MMP-2 with sICAM-1 (P=0.05) and S100beta (P=0.01). The mean MMP-2 levels (in ng/ml) in patients with metastatic melanoma were 196.4±54.0 versus 182.6±46.9 in non-metastasizing melanoma (P=0.037). However, there was no significant difference in MMP-2 levels between the different tumor stages. Conclusion: Determination of MMP-2 serum levels is of limited value as a tumor marker in melanoma, though there are higher levels in the more advanced disease.